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Abstract

The CXCL12/CXCR4 endocrine axis has been demonstrated to play a pivotal role in organ-specific metastasis of many different types of tumors, but the precise role of the CXCL12/CXCR4 autocrine loop remains poorly understood. In this study, we constructed a functional CXCL12/CXCR4 autocrine loop in A549 cells using a gene transfection technique to evaluate its effect on the metastasis of non-small cell lung cancer (NSCLC). Our results demonstrated that the CXCL12/CXCR4 autocrine loop significantly promoted the motility, proliferation and invasiveness of the A549 cells, suggesting a key role of the CXCL12/CXCR4 autocrine loop in NSCLC metastasis. In addition, these findings suggest that targeted therapies directed against CXCR4 should consider the CXCL12 expression status of the NSCLC to be treated, since tumors with autocrine overexpression of CXCL12 may be more suitable for the application of chemokine-based anti-cancer therapies.

Introduction

Chemokines are a large family of small (7–15 kDa),
structurally related heparin-binding proteins that bind to and
activate a family of chemokine receptors. More than 50 chemokines
have been identified and they are classified into 4 families (CXC,
CX3C, CC and C) based on the position of the first two conserved
cysteine residues (1). Chemokine
receptors are 7-transmembrane G protein-coupled receptors. Most are
promiscuous and can bind with high affinity to multiple chemokine
ligands (CXCR, CX3CR, CCR and XCR). At present, 20 chemokine
receptors have been identified.

In normal physiology, chemokines are involved in
proinflammatory and non-inflammatory cell homing by binding to
their cognate receptors (2).
However, increasing evidence implicates these small cytokine-like
proteins and their receptors in tumor biology (3–5).
CXCL12 and its cognate receptor CXCR4 have been shown to regulate
site-specific distant metastasis of many cancer types (6–10).
These studies demonstrated that tumor cells express a high level of
CXCR4 and that tumor metastasis target tissues (lung, liver and
bone) express high levels of the ligand CXCL12, allowing tumor
cells to directionally migrate to target organs via a CXCL12-CXCR4
chemotactic gradient. These studies have led to the current
CXCL12/CXCR4 ‘endocrine axis’ model; CXCR4 expression by metastatic
cells enables those cells to navigate towards organs abundantly
expressing CXCL12. Currently, despite compelling evidence for the
pro-metastatic function of the CXCL12/CXCR4 endocrine axis, little
attention has been devoted to the precise role of the CXCL12/CXCR4
autocrine loop in tumor metastasis.

A previous study reported a pro-metastatic effect of
the CXCL12/CXCR4 autocrine loop when CXCL12 was transfected into
the mammary carcinoma cell line MDA-MB-231 (11). This study also demonstrated that
CXCL12 expression was inversely correlated with disease-free and
overall survival in breast cancer patients. In another study,
increased proliferation was in contrast to reduced metastasis in
vivo, which has been demonstrated as responsible for forced
CXCL12-expressing MDA-MB-231 cells (12). These contradictory results suggest
that the function of the CXCL12/CXCR4 autocrine loop in tumor
growth and metastasis requires further elucidation.

Matrix metalloproteinases (MMPs), which are
multidomain zinc-dependent endopeptidases, are pivotal in cancer
invasion and metastasis (13,14).
Among these MMPs, MMP-2 and MMP-9 have been of particular interest
due to their pathogenic roles in non-small cell lung cancer (NSCLC)
(15–17). Degradation of ECM and basement
membranes by MMP-2 and/or MMP-9 is required for tumor cell invasion
and metastasis (16,18,19).
The correlation between the CXCL12/CXCR4 autocrine loop and the
expression of MMP-2 and MMP-9 remains unknown.

We performed this study to investigate the
previously unknown role of the CXCL12/CXCR4 autocrine loop in cell
growth and distant metastasis of NSCLC. This was achieved by using
a gene transfection technique. Human NSCLC cell line A549, which
does not express endogenous CXCL12, was transfected with
pIRES2-ZsGreen1-CXCL12 or the control vector pIRES2-ZsGreen1 to
establish stable CXCL12 (A549-CXCL12) and control vector
(A549-ZsGreen1) transfectants. In the stable CXCL12-expressing cell
line (A549-CXCL12), a functional CXCL12/CXCR4 autocrine loop was
constructed. The contribution of the CXCL12/CXCR4 autocrine loop
signaling pathway to the migration, proliferation and invasiveness
of A549-CXCL12 cells was evaluated and compared with A549-ZsGreen1
and wild-type A549 cells. We hypothesized that the CXCL12/CXCR4
autocrine loop may stimulate production of MMP-2 and MMP-9.
Therefore, we also investigated whether the CXCL12/CXCR4 autocrine
loop affected MMP-2 and MMP-9 expression in NSCLC cells.

Stable transfection

The human full-length CXCL12 cDNA fragment was
ligated to the cloning site of pIRES2-ZsGreen1, followed by
transformation using One-Shot®E.coli (Invitrogen
Ltd.), verification and amplification. A549 cells were transfected
with either purified or control plasmid using Lipofectamine 2000
reagent (according to the manufacturer’s instructions) followed by
selection with G418. Stable CXCL12 (A549-CXCL12) and control
plasmid (A549-ZsGreen1) transfectants were subsequently established
and verified.

Western blot analyses and reverse
transcription-PCR (RT-PCR) analyses

Proteins in the total cell lysates were resolved by
SDS-PAGE and electrotransferred to a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). After the blot was blocked
in a solution of 4% bovine serum albumin, membrane-bound proteins
were probed overnight with primary antibodies against GAPDH,
CXCL12, CXCR4, MMP-2, MMP-9 or p-ERK. They were then incubated with
horseradish peroxidase-conjugated secondary antibodies for 1 h.
Antibody-bound protein bands were detected with enhanced
chemiluminescence reagents and photographed with Kodak X-OMAT LS
film (Eastman Kodak, Rochester, NY, USA). Quantitative data were
obtained using a computing densitometer and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA, USA).

For RT-PCR analysis, RNA was extracted from total
cell lysates using a TRIzol kit (MDBio, Piscataway, NJ, USA). The
RNA concentration was determined using an ultraviolet
spectrophotometer. The reverse transcription reaction was performed
using 2 μg total RNA that was reverse-transcribed into cDNA
using oligo(dT) primer, then amplified for 30 cycles using 2
oligodeoxynucleotide primers: β-actin sense,
5′-CACGATGGAGGGGCCGGACTCATC-3′ and anti-sense,
5′-TAAAGACCTCTATGCCAACACAGT-3′; CXCL12 sense,
5′-GTCAGCCTGAGCTACAGATGC-3′ and anti-sense,
5′-CTTTAGCTTCGGGTCAATGC-3′; CXCR4 sense, 5′-CCGTGGCAAACTGGTACTTT-3′
and anti-sense, 5′-GACGCCAACATAGACCACCT-3′; MMP-2 sense,
5′-GTGCTGAAGGACACACTAAAGAAGA-3′ and anti-sense,
5′-TTGCCATCCTTCTCAAAGTTGTAGC-3′; or MMP-9 sense,
5′-CACTGTCCACCCCTCAGAGC-3′ and anti-sense,
5′-GCCACTTGTCGGCGATAAGC-3′. PCR was carried out as follows: 94°C
for 4 min, followed by 30 cycles of 94°C for 30 sec, 52°C for 30
sec, 72°C for 25 sec and a final extension for 4 min at 72°C. The
products were visualized on 2% agarose gel after staining with
ethidium bromide.

Wound assay

Cells were grown to confluence in 6-well plates and
starved with serum-free RPMI-1640 medium for 24 h. An injury line
was created with a pipette tip in the center of the dishes.
Following rinsing with phosphate-buffered saline (PBS), cells were
allowed to migrate for 12 h and then photographed (×100). Each
clone was plated and wounded in triplicate and each experiment was
repeated at least 3 times.

Invasion assay

The invasion assay was performed using Transwells
with filter inserts (pore size, 8 μm) coated with 50
μl Matrigel diluted 1:3. Approximately 2.5×104
cells in 100 μl serum-free RPMI-1640 medium were placed in
the upper chamber and 1 ml of the same medium was placed in the
lower chamber. Following 18 h incubation at 37°C in 5%
CO2, non-invading cells were removed from the upper
surface of the filter. Cells that had migrated through the filter
were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA),
stained with crystal violet for Matrigel and counted under a
microscope (×400). Each clone was plated in triplicate in each
experiment, and each experiment performed in triplicate.

MTT assay

Briefly, 1×104 cells were seeded onto
96-well plates and allowed to adhere for 8 h. Cell proliferation
was assessed at various time points by adding 10 μl
filter-sterilized MTT (5 mg/ml in PBS) to a single row of 6 wells.
Following 4 h incubation with MTT, the media was removed and the
blue formazan crystals trapped in the cells were dissolved in 100
μl sterile DMSO, by incubating at 37°C for 30 min.
Absorbance at 568 nm was measured in each well with a plate reader.
The growth curve was constructed by plotting absorbance against
time.

Statistics

Statistical differences between the means of the
different groups were evaluated with Prism 5.01 (GraphPad Software
Inc., La Jolla, CA, USA) using one-way ANOVA. P<0.05 was
considered to indicate a statistically significant difference.

Results

Expression pattern of CXCL12 and CXCR4 in
NSCLC cell lines

Several studies have demonstrated CXCR4 expression
in NSCLC cell lines, but its ligand CXCL12 has not been observed
(10,15,20).
We examined mRNA expression of CXCL12 and CXCR4 in a panel of human
NSCLC cell lines, including 95C, 95D, H1975 and A549, using RT-PCR.
In accordance with previous studies, all 4 cell lines
constitutively expressed CXCR4 mRNA, whereas CXCL12 mRNA expression
was not observed (Fig. 1A).
Following this, total cell lysates of these cell lines were
prepared and examined for CXCL12 and CXCR4 protein expression by
western blot analysis using CXCL12- and CXCR4-specific antibodies.
As demonstrated in Fig. 1B, all 4
cell lines constitutively expressed CXCR4 protein but not its
cognate ligand CXCL12.

The human NSCLC cell line A549 was transfected with
pIRES2-ZsGreen1 plasmid encoding human CXCL12 or empty vector
pIRES2-ZsGreen1 as a control, followed by selection with G418 to
yield a stable CXCL12-expressing cell line (A549-CXCL12) and a
stable control plasmid transfectant (A549-ZsGreen1). Using western
blot analysis, CXCL12 protein expression was observed in
A549-CXCL12 cells, but not in A549-ZsGreen1 or wild-type A549 cells
(Fig. 2A). Furthermore, p-ERK1/2
was detected in A549-CXCL12 cells but not A549-ZsGreen1 cells
(Fig. 2B), indicating autonomous
ERK1/2 activation through CXCL12 binding to its cognate receptor
CXCR4 in A549-CXCL12 cells. Taken together, these data indicated
that a functional CXCL12/CXCR4 autocrine loop was successfully
constructed in the A549-CXCL12 cell line.

To investigate the influence of the CXCL12/CXCR4
autocrine loop on the malignant phenotype of NSCLC cells, we
measured the migration, invasiveness and proliferation of
A549-CXCL12, A549-ZsGreen1 and wild-type A549 cells. In wound
migration analyses, A549-CXCL12 cells demonstrated significantly
increased mobility compared with A549-ZsGreen1 and wild-type A549
cells (P<0.01; Fig. 3A and C).
The invasive potential of A549-CXCL12, A549-ZsGreen1 and wild-type
A549 cells was determined using a Matrigel chamber assay. The
number of invading A549-CXCL12 cells was significantly higher than
(triple) that of A549-ZsGreen1 or wild-type A549 cells (P<0.01;
Fig. 3B and D). The MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell
proliferation assay was performed to measure cell proliferation
rate. As shown by the proliferation curve (Fig. 3E), the cell proliferation rate of
A549-CXCL12 cells was markedly higher than that of A549-ZsGreen1 or
wild-type A549 cells at 24 and 48 h (P<0.01), indicating that
the CXCL12/CXCR4 autocrine loop induced increased cellular
proliferation.

Discussion

Primary non-small cell lung cancer is the leading
cause of cancer mortality worldwide. Most patients present with
locally advanced (37%) or metastatic (38%) disease at the time of
diagnosis (21). As with most
cancers, early-stage NSCLC is often be controlled with locally
directed therapy including radiation and surgery; it is the
development of metastatic disease that leads to the high mortality
rate of NSCLC. Therefore, possible mechanisms of metastasis, as
well as the early detection and screening of lung cancer, have been
the subject of growing interest.

Tumor metastasis is an organized process that occurs
in a stepwise fashion: i) uncontrolled proliferation and local
invasion; ii) intravasation into the vascular system and survival
in the circulation; iii) escape of cancer cells from the lumina of
blood vessels into the parenchyma of distant tissues
(extravasation), and iv) the formation of secondary tumors
(colonization) (22–24). Tumor metastasis is also a
non-random, highly organ-specific pathophysiological process
(23,25,26). A
growing body of evidence has indicated that the chemokine CXCL12
and its cognate receptor CXCR4 are critical in this process
(6,10,27–30).
These studies have led to the current CXCL12/CXCR4 ‘endocrine axis’
model; CXCR4 expression by metastatic cells enables these cells to
navigate towards organs abundantly expressing CXCL12. In the case
of NSCLC, the role of the CXCL12/CXCR4 endocrine axis has been well
established (10), but the precise
effect of the CXCL12/CXCR4 autocrine loop on NSCLC cells has not
yet been demonstrated. Several studies have been conducted to
evaluate the functionality of the CXCL12/CXCR4 autocrine loop in
human mammary carcinoma (11,12),
oral squamous cell carcinoma (31)
and colorectal carcinoma cells (32). However, the evidence arising from
these studies has been controversial and even contradictory.

In this study, CXCR4 was constitutively expressed in
all 4 human NSCLC cell lines evaluated, but CXC12 was not observed
consistently. To investigate the effect of the CXCL12/CXCR4
autocrine loop on the NSCLC cells, the full-length CXCL12
gene was transfected into human NSCLC cell line A549 to generate
the stable CXCL12-expressing transfectant, A549-CXCL12. A549-CXCL12
cells overexpressed CXCL12 and autonomous ERK1/2 activation was
also observed, indicating that this transfectant had acquired a
functional CXCL12/CXCR4 autocrine loop. The ability to migrate was
evaluated first, as this is a significant characteristic of the
aggressive phenotype of malignant tumor cells. Our results
demonstrated that the CXCL12/CXCR4 autocrine loop signaling pathway
significantly enhanced A549-CXCL12 cell migration compared with
control A549-ZsGreen1 and wild-type A549 cells (P<0.01). These
results are consistent with those revealed by Kang et
al(11) and Uchida et
al(31), who performed similar
experiments using human mammary carcinoma cells and oral squamous
cell carcinoma cells, respectively. Metastasis fundamentally
involves the movement of cells from one site to another. These
results indicated that the CXCL12/CXCR4 autocrine loop may promote
the metastatic potential of NSCLC cells.

The most fundamental trait of malignant tumor cells
is their ability to sustain chronic proliferation. Thus, another
significant characteristic of the aggressive phenotype of malignant
tumor cells is uncontrolled cellular proliferation. As a primary
tumor grows, its blood supply cannot support its metabolic needs;
lack of oxygen causes tumor cells to move away from the site of
hypoxia and spread to new locations through activation of genes
such as c-Met and CXCR4(33). Our results revealed that the
CXCL12/CXCR4 autocrine loop induced a significant increase in
A549-CXCL12 cell proliferation compared with the vector control
cell line A549-ZsGreen1 and wild-type parent cell line A549,
indicating that the CXCL12/CXCR4 autocrine loop may play a critical
role in tumor growth so as to facilitate tumor metastasis. These
results are in accordance with studies demonstrating that the
CXCL12/CXCR4 autocrine loop increased mammary carcinoma cell
proliferation (13). However, they
are in disagreement with previous results by Wendt et
al(33), who revealed increased
apoptosis in forced CXCL12-expressing colorectal cancer cells
compared to control eGFP clones. These data highlight differences
in the pathophysiological impact of the CXCL12/CXCR4 autocrine loop
among different types of cancer cells.

In the present study, the CXCL12/CXCR4 autocrine
loop also markedly enhanced the invasiveness of NSCLC cells. Kang
et al and Uchida et al(11,31)
reported that the CXCL12/CXCR4 autocrine loop significantly
increased breast cancer and oral squamous cell carcinoma cell
invasiveness, respectively. By contrast, Wendt et al
reported decreased invasiveness and metastasis in breast cancer
cells and colorectal cancer cells in vivo(12,32),
suggesting that the role of the CXCL12/CXCR4 autocrine loop in
modulating the capacity for invasiveness may depend on the type of
cancer. Enzymatic degradation of ECM and basement membranes is a
key step in cancer invasion and metastasis. In human lung cancer,
MMP-2 and MMP-9 have been demonstrated to be correlated with
malignancy grade and metastasis (13,34).
Therefore, we hypothesized that the CXCL12/CXCR4 autocrine loop may
stimulate MMP-2 and MMP-9 production to degrade the ECM and
basement membranes. Both mRNA and protein expression of MMP-2 and
MMP-9 were markedly increased in A549-CXCL12 cells compared with
controls, further supporting the notion that the CXCL12/CXCR4
autocrine loop indirectly enhanced the capacity for NSCLC cell
invasiveness.

Overall, these results indicate that the
CXCL12/CXCR4 autocrine loop increases the metastatic potential of
NSCLC cells, confirming that the CXCL12/CXCR4 autocrine loop may
contribute to the motility, growth and invasiveness of NSCLC. To
our knowledge, this in vitro study is the first to reveal
that the CXCL12/CXCR4 autocrine loop increases the metastatic
potential of NSCLC. Furthermore, our findings suggest that targeted
therapies against CXCR4 (35)
should consider the CXCL12 expression status of the NSCLC to be
treated, since tumors with autocrine overexpression of CXCL12 may
be more sensitive to CXCR4 antagonists that compete with CXCL12 for
receptor binding and more suitable for the application of
chemokine-based anti-cancer therapies. Differences in the impact of
the CXCL12/CXCR4 autocrine loop on cancer cells may depend on the
type of cancer, and this possibility requires further
investigation.